Cartilage tissue engineering: Strategies to maintain the chondrogenic differentiation potential of culture-expanded mesenchymal progenitor cells

Chantal Voskamp

Research output: Types of ThesisDoctoral ThesisInternal

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Abstract

Focal articular cartilage defects occur often during knee trauma. Articular cartilage has a limited repair capacity, so it is necessary to repair these cartilage defects to prevent further degeneration of the knee joint. Mesenchymal progenitor cells, often referred to as mesenchymal stem or stromal cells (MSCs) are promising cells for cartilage tissue engineering strategies. However, the chondrogenic differentiation capacity of MSCs declines with in vitro expansion. The overall aim of this thesis was to determine how MSCs can preserve their chondrogenic differentiation potential during in vitro expansion.
In chapter 2, we determined how cellular senescence influences the differentiation capacity of MSCs. Therefore, cellular senescence was induced during monolayer and at different time points during chondrogenesis using gamma irradiation. When cellular senescence was induced during expansion or during early chondrogenic differentiation, the cells had a reduced chondrogenic differentiation capacity. When senescence was induced later during chondrogenic differentiation, no significant changes in the chondrogenic markers were observed. To investigate the effect of paracrine senescence, we treated non-senescent pellets with medium conditioned by senescent pellets. After 48 h of exposure, no significant effect on the expression of anabolic or catabolic markers was determined in recipient pellets. Finally, we showed that senescent MSCs had a reduced ability to respond to TGFβ1, one of the key factors to induce chondrogenic differentiation. In conclusion, the results in chapter 2 indicated that the occurrence of cellular senescence in MSCs inhibited early processes of chondrogenic differentiation and thereby the capacity of MSCs to generate cartilage.
High MSC expansion was previously associated with high TWIST1 levels. To better understand how TWIST1 levels affect MSC expansion and senescence, we silenced TWIST1 using siRNAs (chapter 3). Silencing of TWIST1 increased the percentage of senescent MSCs. Surprisingly, TWIST1-silencing-induced senescent MSCs had a non-classical senescence-associated secretory phenotype (SASP) lacking the expression of IL-6 and IL-8, in contrast to irradiation-induced senescent cells. It is known that senescence and their SASP are associated to the metabolic state of the cells. Indeed, when we determined the bioenergetic state, the TWIST1-silencing-induced and irradiation-induced-senescent cells had a different energetic state. Both types of senescent cells had an increased oxygen consumption rate compared to control non-senescent MSCs, but TWIST1-silencing-induced senescent MSCs had a lower extracellular acidification rate, compared to irradiation-induced senescent MSCs. In chapter 4, we used a fluorescent probe-based method (SmartFlare) that has the benefit that it does not require fixation of the cells, allowing to sort living cells based on TWIST1 RNA expression. First, we validated the TWIST1 probe and demonstrated that the probe specifically recognized TWIST1 in MSCs. Next, TWIST1high and TWIST1low expressing MSCs were sorted. TWIST1high expressing MSCs had an increased expansion rate compared to TWIST1low expressing MSCs. In conclusion, the results of chapter 3 and chapter 4 demonstrated that high TWIST1 expressing MSCs had a higher expansion rate compared to low TWIST1 expressing MSCs. Furthermore, using methods to silence TWIST1 we demonstrated that low TWIST1 levels induced cellular senescence in MSCs and that these senescent cells had a specific SASP profile and metabolic state.
In chapter 5, we aimed to determine how different culture methods can influence the expansion and chondrogenic capacity of MSCs. It had been reported that exposure to TNFα, during in vitro expansion, could be potentially beneficial for tissue regeneration. Therefore, we treated the MSCs with TNFα during MSC expansion and showed that the treatment increased the chondrogenic differentiation capacity of the cells. Furthermore, treatment with TNFα during expansion reduced the inhibitory effect of the cytokine during subsequent chondrogenic differentiation. Finally, we show that TNFα pre-treatment increased the levels of SOXC proteins and active β-catenin. In conclusion, the results of chapter 5 revealed that that TNFα pre-treatment could potentially be used as a strategy to improve MSC-based cartilage repair.
To conclude, this thesis showed that different aspects such as 1) low percentage of senescent cells, 2) high expression level of TWIST1, and 3) exposure to TNFα, during the expansion phase of MSCs could be beneficial for their chondrogenic differentiation potential. The knowledge of this thesis can be applied to further improve MSC-based therapies to repair cartilage defects.
Original languageEnglish
Awarding Institution
  • Erasmus University Rotterdam
Supervisors/Advisors
  • van Osch, Gerjo, Supervisor
  • Narcisi, Roberto, Co-supervisor
Award date13 Apr 2023
Place of PublicationRotterdam
Print ISBNs978-94-6458-993-1
Publication statusPublished - 13 Apr 2023

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